Encapsulation of Platinum Prodrugs into PC7A Polymeric Nanoparticles Combined with Immune Checkpoint Inhibitors for Therapeutically Enhanced Multimodal Chemotherapy and Immunotherapy by Activation of the STING Pathway

Abstract Tumor immunotherapy has emerged as one of the most promising therapeutic methods to treat cancer. Despite its clinical application, the immunosuppressive tumor microenvironment compromises the therapeutic efficiency of this technique. To overcome this limitation, many research efforts have been devoted to the development of agents that reprogram the immunosuppressive tumor microenvironment through novel mechanisms. Over the last decade, compounds that intervene through the immunogenic stimulator of interferon genes (STING) pathway have emerged with potential for clinical development. Herein, the encapsulation of chemotherapeutic platinum complexes with a polymer with a cyclic seven‐membered ring (PC7A)‐based polymer into pH‐responsive nanoparticles for multimodal therapeutically enhanced chemotherapy and immunotherapy is presented. This study represents the first nanomaterial with a dual activation mechanism of the STING pathway through DNA fragmentation as well as PC7A binding. The combination of these nanoparticles with immune checkpoint inhibitors demonstrates to nearly fully eradicate a colorectal tumor inside the mouse model by chemotherapy and immunotherapy using the STING pathway.


solution in phosphate-buffered saline) and the cells were further incubated for 4 h. Acidified
SDS solution was then added (100 µL/well) and the plates were kept in the dark for an additional 12 h. Measurements of absorbance were subsequently performed on a Bio-Rad plate reader at 570 nm (peak absorbance) and at 650 nm (background absorbance).

Cell apoptosis assay
CT26 cells were seeded on 6-well plates at a density of 1 × 10 6 cells per well and cultured overnight. The cells were divided into 6 groups for the cytotoxicity study: 1) phosphatebuffered saline, 2) NP1, 3) Oxa, 4) Oxa-C16, and 5) NP2. Group 3, Group 4, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 2 was treated with polymer concentration at 50 μg/mL. After incubation for 48 h, the cells were washed with phosphate-buffered saline, and then stained with Annexin V/Propidium iodide for 15 min.
Finally, the cell apoptosis was detected by flow cytometry, and the data were analyzed by FlowJo software.

Colony formation
CT26 cells were seeded on 6-well plates at a density of 1 × 10 3 cells per well and cultured overnight. The cells were divided into 6 groups for the cytotoxicity study: 1) phosphatebuffered saline, 2) NP1, 3) Oxa, 4) Oxa-C16, and 5) NP2. Group 3, Group 4, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 2 was treated with polymer concentration at 50 μg/mL. The culture media was replaced every two days. After ten days, the cells were fixed with 4% paraformaldehyde and further incubated with 1% crystal violet (Solabio). The colony formation was monitored by optical microscopy.

Live/dead cell staining
CT26 cells were seeded into 6-well plates at a density of 8 × 10 4 cells per well and cultured overnight. The cells were divided into 6 groups for the cytotoxicity study: 1) phosphatebuffered saline, 2) NP1, 3) Oxa, 4) Oxa-C16, and 5) NP2. Group 3, Group 4, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 2 was treated with polymer concentration at 50 μg/mL. After 24 h treatment, the media was removed. The cells were washed with phosphate-buffered saline and then stained with Cell Viability/Cytotoxicity Assay Kit.

Western blot
The CT26 cells were seeded on 6-well plates at a density of 1 × 10 6 cells per well. The cells were divided into 5 groups for WB assays:1) PBS, 2) Oxa, 3) Oxa-C16, 4) NP1, 5) NP2. Group 2, Group 3, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 4 was treated with polymer concentration at 50 μg/mL.
Subsequently, the PVDF films were washed 3 times and incubated with HRP (A0208, Beyotime) conjugated antibodies for 2 h at room temperature. The Western blot images were obtained by Gel imaging system (Tanon 4800, China) with 200 μL of ECL chemiluminescent reagent (KF001, Affinity) added on the top of the membrane. β-Actin (#AF5003, Beyotime) and α-Tubulin (#9099, CST) were employed as protein loading control.

Immunofluorescence characterization using confocal laser scanning microscopy
The cells were seeded onto cover slides at a density of 1 × 10 5 cells per slide and cultured overnight. The cells were divided into 5 groups for immunofluorescence imaging: 1) PBS, 2) Oxa, 3) Oxa-C16, 4) NP1, 5) NP2. Group 2, Group 3, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 4 was treated with polymer concentration at 50 μg/mL. Then, the cells were fixed in a 4% paraformaldehyde solution, 7 blocked with 1% BSA (Beyotime) and incubated with 0.1% Triton (Beyotime). Afterwards, the cells were incubated with P-STING, P-TBK1, P-IRF3, and γ-H2A primary antibody diluted in cell media at 4 ℃ for 12 h. Subsequently, the media was removed and the cells were washed with phosphate-buffered saline. The cells were further incubated with the secondary Alexa Fluor 488-conjugated antibody (ab150077, Abcam) for 2 h. The cell nucleus was stained with DAPI and the cytoskeleton was stained with Phalloidin (Solabio). Images were taken with a confocal laser scanning microscope.

DNA damage evaluation using flow cytometry
The DNA damage in the cancer cells was detected upon monitoring of the DNA damage marker protein γ-H2A. CT26 cells were seeded on 12-well plates at a density of 3 × 10 5 cells per well and cultured overnight. The cells were divided into 5 groups for immunofluorescence imaging: 1) PBS, 2) Oxa, 3) Oxa-C16, 4) NP1, 5) NP2. Group 2, Group 3, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 4 was treated with polymer concentration at 50 μg/mL. After 48 h of incubation, all the cells were fixed in a 4% paraformaldehyde. Afterwards, the cells were incubated with the γ-H2A primary antibody diluted in cell media at 4 ℃ for 12 h. Subsequently, the media was removed and the cells were washed with phosphate-buffered saline. The cells were further incubated with the secondary Alexa Fluor 488-conjugated antibody (ab150077, Abcam) for 2 h. The expression of γ-H2A was then detected via flow cytometry.

ELISA assay
CT26 cells were seeded on 12-well plates at a density of 3 × 10 5 cells per well and cultured overnight. The cells were divided into 5 groups for ELISA: 1) PBS, 2) Oxa, 3) Oxa-C16, 4) NP1, 5) NP2. Group 2, Group 3, and Group 5 were treated with the indicated drugs at a Pt concentration of 23.25 μM, and Group 4 was treated with polymer concentration at 50 μg/mL. Subsequently, the media was removed and the cells were washed with phosphate-buffered saline. The cells were centrifuged at 12000 rpm/min for 5 min. The obtained supernatant was then detected using a mouse IL-6 ELISA kit (EK2236-01, Multi Sciences) and a mouse IFN-β ELISA kit (EK206HS-02, Multi Sciences) according to the manufacturers protocol.

Maturation of bone-marrow derived dendritic cells
Bone-marrow derived dendritic cells were obtained from 5 to 6 week-old female C57BL/6 mice and cultured in RPMI 1640 medium supplement with 10% FBS, granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL, Beyond), and interleukin-4 (IL-4) (10 ng/mL, CT26 cells were seeded on 12-well plates at a density of 3 × 10 5 cells per well. After 24 h, the cells were treated with Oxa, Oxa-C16, NP1, or NP2 for 48 h. The CT26 cells were incubated with the obtained bone-marrow derived dendritic cells for 24 h. Subsequently, the dendritic cells were stained with the corresponding antibody (anti-CD11c-PE, anti-CD80-FITC, and anti-CD86-APC) for 1 h. The maturation of the dendritic cells was assessed by flow cytometry.

Metabolomics assay
The cells were seeded on the cell culture plates and cultured overnight. After 24 h, the media was removed and the cells were treated with Oxa, Oxa-C16, NP1, or NP2 for 48 h. For Pt containing group, the concentration for the treatment was fixed at 23.25 μM. For NP1, the concentration for the polymer was 50 μg/mL. Then, the cells were washed with phosphate-

In vivo biocompatibility evaluation
For the biocompatibility study, healthy Kunming mice were randomly divided into 6 groups: 1) PBS, 2) Oxa, 3) NP1, 4) anti PD-L1, 5) NP2, and 6) NP2 + anti PD-L1. Group 2, Group 5, and Group 6 were intravenously injected with drugs at a Pt dose of 3 mg/kg. Group 4 and Group 6 were intraperitoneal injected with anti PD-L1 at a dose of 5 mg/kg. The body weight of mice was monitored during 15 days treatment. Finally, the major organs were 9 collected and a slice of each one was fixed with 4% paraformaldehyde. The obtained slices were stained with hematoxylin and eosin.

Hemolysis study
For the hemolysis study, blood samples were obtained from the mice. The blood was centrifuged at 200 rpm for 5 min. The obtained blood serum was incubated with PBS, Oxa, Oxa-C16, NP1, NP2 at 37 ℃ for 3 h. For Oxa, Oxa-C16, NP2, the samples were treated with the indicated drugs at a Pt concentration of 300 μM. For NP1, the serum was treated with polymer concentration at 500 μg/mL. The absorption at 541 nm was measured with a microplate reader. The hemolysis ratio was calculated using the following formula: DT-experimental group, DNC-negative control group, DPC-positive control group

Biodistribution study
The CT26 tumor bearing mice were administrated with NP2@Cy7.5 via intravenously injection. The fluorescence imaging proceeded using an IVIS system (Spectrum CT, PerkinElmer) at various time intervals. The in vivo biodistribution of NP2@Cy7.5 was observed on the Cy7.5 channel (λex= 745 nm, λem= 840 nm). At 72 h-post injection, the mice were sacrificed, and their organs were harvested and imaged ex vivo using the same parameters.
The average photon flux in radians for the different reporter signals in each excised organ were quantified.

Antitumor efficacy study
CT26 cells (3 × 10 6 ) were subcutaneously injected into the right hip of female BALB/c mice. When the size of tumor reached 100 mm 3 , the mice were randomly divided into 6 groups (5 mice in each group), which includes: 1) PBS, 2) Oxa, 3) NP1, 4) anti PD-L1, 5) NP2, and 6) NP2 + anti PD-L1. Group 2, Group 5, and Group 6 were intravenously injected with drugs at a Pt dose of 3 mg/kg. Group 4 and Group 6 were intraperitoneal injected with anti PD-L1 at a dose of 5 mg/kg. The body weight and tumor volume were recorded every two days. The tumor volume was calculated using the following equation: Tumor volume = 1/2×L×W 2 , where "L" is the long diameter of the tumor, and "W" is the short diameter of the tumor. Data are presented as means ± SD (n =5).

Maturation of dendritic cells in CT26 xenograft mouse model
The peripheral blood was collected from mice treated with different drugs for ELISA tests through centrifuging at a speed of 12000 rpm/min for 5 min. The animal tissues were accurately weighed and 9 times the volume of homogenizing medium (0.86% or 0.9% normal saline is recommended) was added at the ratio of weight (mg) : volume (µl) = 1:9. Under the condition of ice water bath, the homogenization was performed mechanically to prepare 10% homogenate, and the supernatant was centrifuged for 10 minutes from 2500 to 3000 rpm. The above peripheral blood was prepared for cytokines analysis using Mouse IL-6 High Sensitivity ELISA kits, Mouse IFN-β ELISA Kit and Mouse IFN-γ High Sensitivity ELISA kits (EK280HS-01, Multi Sciences). The p-values were found to the following range: ns = no statistical difference, * p < 0.05, **p < 0.01, ***p < 0.001.